(I-348) Measurement of Cationic Solute Diffusion and Fixed Charge Density in Human Synovium

Friday, October 13, 2023

3:30 PM – 4:30 PM PDT

Location: Exhibit Hall - Row I - Poster # 348

Presenting Author(s)

AD

Alexandra Davis (she/her/hers)

Graduate Student
Washington University in St. Louis
St. Louis, Missouri, United States

Co-Author(s)

AV

Ashish Vaidyanathan

Undergraduate
Washington University in St. Louis, United States
MR

Milad Rohanifar

Senior Analytical Engineer
American Axle & Manufacturing, United States

Primary Investigator(s)

LS

Lori Setton

Lucy & Stanley Lopata Distinguished Professor of Biomedical Engineering
Washington University in St. Louis, United States

Introduction:: The synovium is a thin, multilayer tissue that lines diarthrodial joints. Its blood and lymphatic vessels rapidly clear small and large molecules, respectively, from the joint space and therefore limit the efficacy of intra-articular (IA) drug delivery to treat degenerative joint diseases such as arthritis [1,2]. In vivo pharmacological studies may overestimate drug clearance times because drug concentrations are typically measured in large volumes of blood; the geometry of the knee, such as synovial fluid volume and surface area, could also differ between species and contribute to varying drug half-lives in vivo. However, experimental-computational approaches can be used to quantify intrinsic solute diffusivities, enabling comparisons across species, solutes, and pathologies. Previously, we built a multiphasic finite element model (FEM) to measure diffusion through synovium and found that the effective diffusivity (Deff) of an uncharged solute decreased with increasing molecular weight (MW) [3,4]. Factors other than MW could affect solute diffusion through synovium, as the synovial matrix contains charged species such as collagen and glycosaminoglycans (GAGs). Thus, this study aimed to quantify the Deff of a cationic solute through human synovium in vitro as well as the fixed charge density (FCD) of synovium for the first time.

Materials and Methods:: Human synovium was harvested from donor knee tissues and prepared as previously described [4]. FCD quantification: A 1,9-dimethylmethylene blue (DMMB) assay was performed as previously described [5] using a standard curve of chondroitin sulfate (MW 502.5 g/mol) to measure the concentrations of sulfated GAG (sGAG) in synovium (n=4), which were used to calculate FCD assuming two moles of charge per sGAG. Diffusion experiment: Diffusion of cationic 4-kDa fluorescein isothiocyanate-diethylaminoethyl-dextran (FITC-DEAE) (TdB Labs) through synovium (n=9) was studied with a custom-built diffusion chamber as previously described [4]. The zeta potential of the charged solute was measured (ZEN3600, Malvern Instruments) to confirm its positive charge. Model predictions of unsteady diffusion: FEBio was used to model unsteady diffusion of FITC-DEAE through synovium [6], which was represented as a porous, fluid-saturated, and uncharged mixture based on the negligible FCD from the DMMB results. The solid phase was assumed to be neo-Hookean and assigned previously measured material properties [7]. Electroneutrality was satisfied by introducing an anionic counter-ion for the first 100s of the simulation. Specimen-specific meshes of thickness L contained eight-node hexahedral elements refined at the interface between the donor bath and synovium (Fig. 1a). A single Deff was estimated for each sample through numerical matching of experimental concentration data to FEBio model predictions. Statistics: The Mann-Whitney U Test was performed to determine if the Deff of FITC-DEAE through human synovium significantly differed (p< 0.05) from that of 3-kDa Texas Red dextran measured in our previous study.

Results, Conclusions, and Discussions::

Human synovium was found to contain very low concentrations of sGAG (0.112 ± 0.0213 mg/g synovium) and was estimated to have a negligible FCD (−0.013 ± 0.0025 mM). Thus, the synovium was assigned a FCD of zero in FEBio to model charged solute transport. The zeta potential of FITC-DEAE was +1.69 ± 0.342 mV. The FEM predictions of charged solute transport agreed well with the experimental data according to nonlinear regression fit statistics (RSS < 0.03). The Deff of 4-kDa FITC-DEAE (1.76E-04 ± 5.90E-05 mm2/s) through human synovium was not significantly different (p=0.0755) from that of neutral 3-kDa Texas Red (9.73E-05 ± 2.66E-05 mm2/s) measured in our previous study (Fig. 1b).

The results of the diffusion study suggest that cationic charge does not significantly affect diffusivity of low MW solutes through synovium. Here, human synovium showed a low sGAG concentration that agrees with prior studies [10,11] and a FCD orders of magnitude lower than that of other soft tissues [8,9]. This may explain why cationic and neutral dextrans diffused similarly through synovium. However, sGAG concentration may decrease with age and should be measured in tissues from younger donors to clarify how FCD affects solute transport. Although the DMMB assay cannot detect hyaluronic acid (HA), an unsulfated GAG produced by synovial fibroblasts, HA likely does not contribute large amounts of negative charge in the matrix [10]. Instead, HA could elicit a boundary-layer effect at the synovial intima and impede solute transport through a different mechanism. The effect of charge on diffusivity of larger solutes as well as the role of in vivo factors such as cell-mediated uptake, IA pressure, and the density of draining lymphatic vessels should be explored in future work. Ultimately, these results inform further studies on charged solute-matrix interactions in synovium to guide drug design for IA drug delivery.

Acknowledgements (Optional): : This work was funded by NIH AR070975 and AR077678. We thank Mid-America Transplant for providing human donor knee tissues

References (Optional): :

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